Incretin-Based Multi-Agonist Peptides Are Neuroprotective and Anti-Inflammatory in Cellular Models of Neurodegeneration

Glucagon-like peptide-1 (GLP-1)-based drugs have been approved by the United States Food and Drug Administration (FDA) and are widely used to treat type 2 diabetes mellitus (T2DM) and obesity. More recent developments of unimolecular peptides targeting multiple incretin-related receptors (“multi-agonists”), including the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) and the glucagon (Gcg) receptor (GcgR), have emerged with the aim of enhancing drug benefits. In this study, we utilized human and mouse microglial cell lines, HMC3 and IMG, respectively, together with the human neuroblastoma SH-SY5Y cell line as cellular models of neurodegeneration. Using these cell lines, we studied the neuroprotective and anti-inflammatory capacity of several multi-agonists in comparison with a single GLP-1 receptor (GLP-1R) agonist, exendin-4. Our data demonstrate that the two selected GLP-1R/GIPR dual agonists and a GLP-1R/GIPR/GcgR triple agonist not only have neurotrophic and neuroprotective effects but also have anti-neuroinflammatory properties, as indicated by the decreased microglial cyclooxygenase 2 (COX2) expression, nitrite production, and pro-inflammatory cytokine release. In addition, our results indicate that these multi-agonists have the potential to outperform commercially available single GLP-1R agonists in neurodegenerative disease treatment.


Introduction
Significant scientific, medical, and media attention has been given to incretin mimetics in the treatment of type 2 diabetes mellitus (T2DM) and obesity, as well as in numerous additional seemingly unrelated conditions-and rightfully so.Glucagon-like peptide-1 (GLP-1) and, more recently, glucose-dependent insulinotropic polypeptide (GIP) and glucagon (Gcg) receptor (R) agonists have revolutionized T2DM and obesity treatment, restoring insulin sensitivity and often facilitating weight loss in patients [1,2].Moreover, current research suggests the potential for repurposing this drug class to treat various additional diseases, including prostate cancer [3], polycystic ovary syndrome [4], substance abuse [5], and diseases/disorders of the brain with an inflammatory component, such as traumatic brain injury (TBI) [6,7], ischemic stroke [8,9], idiopathic intracranial hypertension [10], and neurodegenerative diseases including Alzheimer's (AD) [11,12] and Parkinson's diseases (PD) [8,11,13].Given the insufficiency of existing treatment options for these disorders, particularly AD and PD, repurposing already United States Food and Drug Administration (FDA)-approved incretin mimetics could yield a novel therapeutic strategy to more efficiently meet the urgent need to treat neurodegenerative diseases.
In the human central nervous system (CNS), incretin mimetics have the potential to enact insulinotropic, neuroprotective, antiapoptotic, antioxidative, and anti-inflammatory benefits-pleiotropic mechanisms of action that can afford various anti-neurodegenerative protections.Clinical trials investigating commercially available GLP-1R agonists as treatments for PD and AD have already been completed, with encouraging results comprising improved off-medication motor scores, motor ability, and cognitive function in PD patients [40][41][42][43], as well as improved brain glucose metabolism [44] and reduced amyloid-β 1-42 concentration in neuronal-derived extracellular vesicles [45] in AD patients.Numerous ongoing clinical trials are investigating various GLP-1R agonist analogs, including exenatide, liraglutide, semaglutide, and lixisenatide, as anti-neurodegenerative agents in AD and PD patients [46,47].
Recently, the development of unimolecular incretin receptor multi-agonists has received much attention in the T2DM field.Tirzepatide/Mounjaro ® , a novel GLP-1R/GIPR dual agonist, was recently approved by the FDA for T2DM treatment, and demonstrated enhanced insulinotropic benefits relative to single GLP-1R agonists in clinical trials [48].The development and evaluation of additional GLP-1R/GIPR dual and GLP-1R/GIPR/GcgR triple agonists are well underway for T2DM treatment, but studies in repurposing them for neurodegenerative disorders lag behind.In this work, we aim to aid the evaluation of these novel incretin receptor multi-agonists in preclinical models of neurodegeneration and thereby inform potential future clinical trials.We highlight promising findings in cell culture models of neurodegeneration indicating the enhanced neuroprotective, antioxidative, and anti-inflammatory benefits of the three selected multi-agonists-the dual GLP-1R/GIPR agonists "Twincretin" and LY329 (known commercially as Tirzepatide/Mounjaro ® ) and the triple GLP-1R/GIPR/GcgR agonist "Triagonist" (known clinically as Retatrutide)-relative to a single GLP-1R agonist, exendin-4 (known clinically as Exenatide).

Cell Culture
All cell line cultures were maintained in a 37 • C incubator comprising 5% CO 2 and 95% air, with medium replacement every other day.

Drug and Challenge Dose Selection
The selection of an incretin mimetic dose for evaluation in neurotrophic, neuroprotective, and inflammatory studies across SH-SY5Y, HMC3, and IMG cells was based on pilot dose-response studies that were focused on identifying the threshold concentration to provide the biological action of interest, and this dose was then used across single, dual, and triple receptor agonists to allow their side-by-side comparison at an equimolar concentration.Likewise, for the selection of a physiological/drug-induced (i.e., H 2 O 2 , LPS, STS) challenge, concentrations were selected from prior pilot dose-response studies focused to achieve a biologically significant but sub-maximal effect.

Cellular ROS Assay
The Fluorescent DCFDA Cellular Reactive Oxygen Species (ROS) Detection Assay was obtained from Abcam (ab113851, Waltham, Boston, MA, USA).SH-SY5Y and HMC3 cells were seeded at a density of 2.5 × 10 4 cells per well the day before treatment.To induce oxidative stress in both cell lines, we used a 1 h H 2 O 2 treatment.Prior to the H 2 O 2 challenge, cells were pre-treated with 100 nM incretin-based peptide or vehicle for 2 h.Cellular ROS were measured with the assay kit in accordance with the manufacturer's protocol.Assay plates were read using an Infinite m200 Pro plate reader with i-Control software (Tecan).

Cytokine ELISAs
IMG cells were seeded in 24-well dishes at a density of 2.0 × 10 5 cells per well.Following a 24 h 100 nM incretin-based peptide pre-treatment and a 24 h 1 ng/mL LPS pro-inflammatory stimulus, cell media was collected for use in cytokine and prostaglandin enzyme-linked immunosorbent assays (ELISAs), as well as in Griess nitrite assays.Mouse interleukin (IL)-6 and mouse tumor necrosis factor-alpha (TNF-α) levels in cell culture media samples were measured utilizing BioLegend's corresponding ELISA MAX™ Deluxe Sets (431315 and 430915, San Diego, CA, USA) in accordance with the manufacturer's protocol.Assay plates were read using a SpectraMax Plus 384 Microplate Reader with SoftMax ® Pro 7 software (Molecular Devices, Sunnyvale, CA, USA).

Prostaglandin E 2 ELISAs
The Prostaglandin E 2 ELISA Kit-Monoclonal was purchased from Cayman Chemical (514010, Ann Arbor, MI, USA) and utilized to quantify prostaglandin E 2 (PGE2) levels in undiluted cell culture media samples obtained as indicated above in accordance with the manufacturer's protocol.Assay plates were read using an Infinite m200 Pro plate reader with i-Control software (Tecan).

Nitrite Detection Assay
The Griess Reagent System, a colorimetric nitrite assay kit, was purchased from Promega (G2930) and utilized to quantify nitrite levels in undiluted cell culture media samples obtained as indicated above in accordance with the manufacturer's protocol.Assay plates were read using a SpectraMax Plus 384 Microplate Reader with SoftMax ® Pro 7 software (Molecular Devices).

Caspase-3 Activity Assay
The Fluorimetric Caspase-3 Assay Kit was purchased from Sigma (CASP3F-1KT, Saint Louis, MO, USA).SH-SY5Y cells were seeded in 24-well plates at a density of 1.25 × 10 5 cells per well the day before treatment.Cells were first pre-treated with incretinbased peptide for 24 h, followed by the addition of STS to its final concentration for another 24 h.At the end of treatment, cells were collected in lysis buffer and spun at 10,000× g for 5 min to remove debris.Supernatant was used in the caspase-3 activity assay as well as a BCA assay for the measurement of protein content and used in accordance with the manufacturer's protocol.Assay plates were read using an Infinite m200 Pro plate reader with i-Control software (Tecan), and the final caspase-3 activity results were normalized to protein content in the samples.

Western Blot
IMG cells were seeded in 60 mm dishes at a density of 2 × 10 6 cells per dish.After a 24 h 100 nM incretin-based peptide pre-treatment and a 24 h 1 ng/mL LPS pro-inflammatory stimulus, cells were harvested using a cell scrape, collected by centrifugation, and resuspended in RIPA buffer (R0278, Sigma-Aldrich) with 1X Halt TM Protease & Phosphatase Single-Use Inhibitor Cocktail (78442, ThermoFisher Scientific, Waltham, MA, USA) for lysis.Samples were vortexed heavily every 5 min for 30 min and centrifuged at the end of the lysis period.Supernatant was collected for Western blotting and the Pierce BCA Protein Assay (23225, ThermoFisher Scientific) to determine total protein concentration.

Statistical Analysis
Results are provided as the mean ± standard error of the mean values throughout.Statistical analyses, detailed in the legend of each figure, consisted of one-way ANOVA with either Dunnett's or Tukey's multiple comparisons test depending on the experiment, all with a significance threshold of α = 0.05.Statistical analyses were performed using GraphPad Prism software (GraphPad Prism 10.2.3, GraphPad Software, Boston, MA, USA).

Trophic Properties of Exendin-4 and Multi-Agonists in Neuronal and Microglial Cell Lines
Our research group and others have established that single GLP-1R agonists can promote neurotrophic effects in human neuronal SH-SY5Y cells [30,[51][52][53][54].We now seek to compare the trophic abilities of the dual GLP-1R/GIPR agonists Twincretin and LY329 as well as the triple GLP-1R/GIPR/GcgR agonist Triagonist with those of the single GLP-1R agonist exendin-4 in SH-SY5Y cells as well as two microglial cell lines (Figure 1).To this end, we treated SH-SY5Y cells with 10 nM exendin-4, Twincretin, LY329, or Triagonist for 48 h and compared cell viability via MTS assays (Figure 1A).Each incretin-based peptide significantly enhanced SH-SY5Y viability relative to the untreated control (exendin-4 p < 0.0001, Twincretin p < 0.01, LY329 p < 0.05, Triagonist p < 0.0001).We performed a similar study in human microglial HMC3 cells-cells were treated with a 10 nM concentration of each incretin peptide and viability was determined via MTS assays after 48 h (Figure 1B).Only the dual GLP-1R/GIPR agonists, Twincretin and LY329, significantly enhanced HMC3 viability (p < 0.01).In contrast, in mouse IMG microglial cells treated for 48 h with 100 nM of each drug, exendin-4 (p < 0.001) and all multi-agonists (p < 0.0001) significantly enhanced IMG cell viability relative to the control (Figure 1C), with the multi-agonists enhancing it to a greater degree than exendin-4 (p < 0.0001).These incretin-based peptide concentrations were specifically selected from pilot study dose-response curves in each cell line focused on defining the threshold activity of the peptides.We have previously shown the neuroprotective potential of incretin-based single and multi-agonists against a variety of cellular insults and CNS injuries in both cell culture and animal models of neurodegeneration [7,8,51,55,56].In the present study, we directly compare the neuroprotective potential of single GLP-1R agonist exendin-4 vs. that of dual and triple incretin-based receptor agonists in both microglial and neuronal cell lines (Figure 2A,B).For HMC3 (Figure 2A) and SH-SY5Y (Figure 2B) cell viability assays, cells were pre-treated with vehicle or 10 nM exendin-4, Twincretin, LY329, or Triagonist for 24 h; then, a 400 µM H 2 O 2 challenge-determined via pilot dose-response studies to achieve 25-50% cell death-was added for another 24 h to stimulate oxidative stress and neurotoxicity.Cell viability was determined via MTS assay.Under this condition, the multi-agonists Twincretin, LY329, and Triagonist significantly enhanced SH-SY5Y viability relative to the H 2 O 2 challenge alone (p < 0.0001), whereas exendin-4 did not (Figure 2B).LY329 (p < 0.01) and Triagonist (p < 0.001) treatments significantly enhanced SH-SY5Y viability relative to exendin-4 treatment (Figure 2B).In HMC3 cells, all multi-agonists (p < 0.0001) and the single agonist exendin-4 (p < 0.01) significantly enhanced viability in the presence of H 2 O 2 , with Twincretin (p < 0.0001) and LY329 (p < 0.05) significantly outperforming exendin-4 (Figure 2A).
Our previous work additionally found that the GLP-1 metabolite GLP-1 (9-36) and exendin-4 were effective in significantly reducing ROS in SH-SY5Y cells [51]; we now test whether our multi-agonists of interest demonstrate similar antioxidative properties (Figure 2C,D).HMC3 microglial cells (Figure 2C) were pre-treated for 2 h with the vehicle or 100 nM GLP-1, Twincretin, LY329, or Triagonist, then exposed to either the vehicle or 150 µM H 2 O 2 for 1 h.The 150 µM H 2 O 2 concentration was determined in prior doseresponse studies to achieve an approximate 50% elevation in ROS; the 100 nM incretinbased peptide concentration was selected from pilot study dose-response data focused on defining threshold activity.ROS levels were determined by a DCFDA assay.In the absence of H 2 O 2 , the multi-agonists Twincretin, LY329, and Triagonist significantly reduced basal ROS levels in HMC3 cells (Figure 2C; Twincretin p < 0.01, LY329 p < 0.0001, Triagonist p < 0.05); however, with H 2 O 2 challenge, no significant incretin antioxidant effects were observed in HMC3 cells.SH-SY5Y neuronal cells (Figure 2D

Exendin-4 and Multi-Agonists Are Antiapoptotic in STS-Challenged SH-SY5Y Neurons
Neuronal and glial apoptosis is a driving feature of cognitive and physical impairment in neurodegenerative disease.In order to study the potential antiapoptotic effects of our incretin-based multi-agonists, we employed STS, a compound known to initiate apoptosis via caspase-3-dependent and -independent mechanisms [57], in human neuronal SH-SY5Y cells to simulate pro-apoptotic and neurotoxic conditions of the neurodegenerative disease brain.We optimized the STS dose and treatment time to achieve 50-150% elevations in cytotoxicity.Cells were first pre-treated with 100 nM concentrations of the single agonist exendin-4 (Figure 3A), the dual agonist Twincretin (Figure 3B), or the triple agonist Triagonist (Figure 3C) for 24 h, then were exposed to a 100 or 250 nM STS challenge for an additional 24 h.These incretin and STS doses were selected from prior pilot dose-response curves.Cytotoxicity, as measured by the CytoTox-ONE™ lactate dehydrogenase (LDH) assay, was dose-dependently elevated by STS, and was significantly reduced in both 100 and 250 nM STS conditions by exendin-4 (Figure 3A, p < 0.05) and by Twincretin (Figure 3B, p < 0.01).Triagonist significantly reduced cytotoxicity only in the 250 nM STS condition (Figure 3C, p < 0.0001).
To further investigate cellular mechanisms behind this observed antiapoptotic activity, we quantified caspase-3 activity in STS-challenged SH-SY5Y cells treated with our incretin-based peptides of interest.SH-SY5Y cells were pre-treated for 24 h with 100 nM concentrations of exendin-4, Twincretin, or Triagonist and were then challenged with 250 nM STS for 24 h.Interestingly, only the Triagonist treatment significantly reduced caspase-3 activity relative to STS challenge alone (Figure 4), suggesting that perhaps the triple agonist we used in this study may be superior to single or dual agonists in terms of reducing caspase-3 activity under this condition.

Exendin-4 and Multi-Agonists Are Antiapoptotic in STS-Challenged SH-SY5Y Neurons
Neuronal and glial apoptosis is a driving feature of cognitive and physical impairment in neurodegenerative disease.In order to study the potential antiapoptotic effects of our incretin-based multi-agonists, we employed STS, a compound known to initiate apoptosis via caspase-3-dependent and -independent mechanisms [57], in human neuronal SH-SY5Y cells to simulate pro-apoptotic and neurotoxic conditions of the neurodegenerative disease brain.We optimized the STS dose and treatment time to achieve 50-150% elevations in cytotoxicity.Cells were first pre-treated with 100 nM concentrations of the single agonist exendin-4 (Figure 3A), the dual agonist Twincretin (Figure 3B), or the triple agonist Triagonist (Figure 3C) for 24 h, then were exposed to a 100 or 250 nM STS challenge for an additional 24 h.These incretin and STS doses were selected from prior pilot doseresponse curves.Cytotoxicity, as measured by the CytoTox-ONE™ lactate dehydrogenase (LDH) assay, was dose-dependently elevated by STS, and was significantly reduced in both 100 and 250 nM STS conditions by exendin-4 (Figure 3A, p < 0.05) and by Twincretin (Figure 3B, p < 0.01).Triagonist significantly reduced cytotoxicity only in the 250 nM STS condition (Figure 3C, p < 0.0001).To further investigate cellular mechanisms behind this observed antiapoptotic activity, we quantified caspase-3 activity in STS-challenged SH-SY5Y cells treated with our incretin-based peptides of interest.SH-SY5Y cells were pre-treated for 24 h with 100 nM concentrations of exendin-4, Twincretin, or Triagonist and were then challenged with 250 nM STS for 24 h.Interestingly, only the Triagonist treatment significantly reduced caspase-3 activity relative to STS challenge alone (Figure 4), suggesting that perhaps the triple agonist we used in this study may be superior to single or dual agonists in terms of reducing caspase-3 activity under this condition.

Multi-Agonists Dampen Neuroinflammatory Signaling of LPS-Stimulated IMG Cells
To study the potential for incretin receptor multi-agonists to mitigate neuroinflammation as an additional anti-neurodegenerative mechanism of action, we utilized mouse IMG cells challenged with LPS as a cellular model of neuroinflammation.IMG cells have been established as a useful microglial cell line for studies of neuroinflammation [50,58], and also have been shown to express GLP-1R and other incretin receptors [51].LPS, a membrane component of Gram-negative bacteria, binds to Toll-like receptor 4 (TLR4) and stimulates downstream signaling pathways, culminating in microglial production and the release of inflammatory and neurotoxic factors, including pro-inflammatory cytokines, chemokines, and prostaglandins [59,60].In early optimization experiments, we tested various doses and treatment times of LPS in IMG cells and found that 1 ng/mL LPS for 24 h produced a consistent elevation in inflammatory markers within detection range of our assays.
As in our previous experiments with HMC3 and SH-SY5Y cells, IMG cells were pretreated for 24 h with the vehicle or 100 nM concentrations of exendin-4, Twincretin, LY329, or Triagonist.Cells were then exposed to a pro-inflammatory challenge of 1 ng/mL LPS for 24 h.For cytokine, PGE2, and nitrite quantification, media was collected after the treatment period for use in TNF-α, IL-6, or PGE2 ELISAs and in Griess reagent system nitrite assays.Cell viability was determined via MTS assay at this stage for inflammatory marker normalization.For COX2 Western blot studies, cell lysates were collected at the end of the treatment period, and COX2 bands were normalized to housekeeping protein GAPDH.Similar to prior experiments, drug and LPS doses were selected from prior pilot dose-response studies.
Oxidative stress and ROS abundance drive neuronal and glial cell death, mitochondrial dysfunction, and inflammation in neurodegenerative disease [77,78], thus emphasizing the utility of drugs targeting oxidative stress to treat neurodegeneration.In our study, we found that incretin-based multi-agonists have significant neuroprotective and antioxidative capacity against a H 2 O 2 -induced oxidative stress challenge in both neuronal SH-SY5Y and microglial HMC3 cells.Of note, the dual and triple agonists demonstrated enhanced antioxidative and neuroprotective effects, as indicated by elevations in cell viability in the presence of H 2 O 2 and reductions in cellular ROS relative to exendin-4, with some significant differences found between multi-agonist-treated groups and the exendin-4-treated group (Figure 2).Previous works in cellular and animal models of neurodegeneration have highlighted enhanced neuroprotective and antioxidative benefits of GLP-1R/GIPR dual agonists [75,79,80] and GLP-1R/GIPR/GcgR triple agonists [7,81] relative to single GLP-1R agonists, reinforcing our current findings.Mechanistically, downstream of GLP-1R/GIPR/GcgR activation, antioxidative signaling driven by Epac and APE1 may be involved in protecting cells and organisms against oxidative stress and oxidative stressinduced neurodegeneration (Figure 6).
Caspase-3-dependent and -independent mechanisms of apoptosis additionally play a role in neuronal death in neurodegenerative disease.In our experiments, we utilized an established pro-apoptotic agent, STS, to stimulate apoptosis in neuronal SH-SY5Y cells and evaluated whether our incretin-based dual and triple agonists could protect cells against apoptosis.We quantified cytotoxicity following treatment and observed that the single agonist exendin-4, the dual agonist Twincretin, and the triple agonist Triagonist reduced SH-SY5Y cytotoxicity induced by 100 nM and/or 250 nM STS with increasing degrees of significance (Figure 3).Interestingly, in a parallel experiment measuring caspase-3 activity, only Triagonist significantly reduced caspase-3 activity (Figure 4), suggesting that STS and incretin-based peptides may interact with apoptotic pathways through both caspase-3dependent and -independent mechanisms, perhaps involving the Bcl-2/Bcl-xL-mediated inhibition of apoptotic executioners and/or the Akt-mediated inhibition of pro-apoptotic proteins (Figure 6).Indeed, other groups have found that dual GLP-1R/GIPR agonists reduced the Bax (pro-apoptotic) to Bcl2 (antiapoptotic) ratio as well as increasing the levels of phosphorylated Akt in cortical and hippocampal cells derived from rat stroke and AD models [82,83], with the dual agonist providing greater antiapoptotic protections than a GLP-1R single agonist [82].Future research could investigate the relative contributions of various antiapoptotic signaling pathways implicated in GLP-1R/GIPR/GcgR antiapoptotic activity, as well as the relative contributions of GLP-1R, GIPR, and GcgR stimulation in mitigating apoptosis.
It has been well established in mammalian cells that LPS induces inflammation primarily through TLR4 stimulation.A recent paper demonstrated that central neuronal GLP-1R activity plays an essential role in combatting TLR4-mediated inflammation [90].Further, the anti-inflammatory effects of single GLP-1R agonist exendin-4 have been previously reported in RAW264.7 macrophages challenged with LPS [59].Here, we demonstrate for the first time that in microglial cells, in addition to exendin-4, incretin-based multi-agonists can significantly reduce multiple LPS-induced inflammation mediators, such as nitrite, COX2, PGE2, and pro-inflammatory cytokines TNF-α and IL-6, in line with previous findings [59].
Our results in IMG cells indicate that multi-agonists could elicit anti-inflammatory effects directly through GLP-1R activity, as well as through GIPR or GcgR activity, in microglia.Although most existing studies of the neuroprotective and anti-inflammatory activity of incretin-based compounds focus on GLP-1R-mediated benefits, we and others have shown that the activation of GIPR has similar effects [91,92].In addition, our previous study of Triagonist in SH-SY5Y cells has shown that each receptor (GLP-1R, GIPR, and GcgR) contributed to the drug's demonstrated neuroprotective effects [7], further emphasizing the possible advantages of multi-agonism over single GLP-1R agonism in the treatment of various neurological disorders.
Our results reproduce the superior anti-inflammatory effects of incretin-based multiagonists compared to single GLP-1R agonists tested in previous preclinical studies.In a mouse model of PD, a novel GLP-1R/GIPR dual agonist outperformed single GLP-1R agonist NLY01 in reducing the levels of activated NF-κB, TNF-α, IL-6, and IL-1β, as well as mitigating microglial activation and reactive astrogliosis [93].Further, a mouse study of AD found that treatment with a dual GLP-1R/GIPR agonist significantly reduced reactive astrogliosis relative to treatment with the single GLP-1R agonist liraglutide [80].Our group has previously demonstrated Triagonist to induce cAMP significantly more than exendin-4 in SH-SY5Y cells, and to reduce TNF-α against an LPS challenge in primary mouse microglia [7].Similarly, a triple GLP-1R/GIPR/GcgR agonist was found to impede reactive astrogliosis and microglial pro-inflammatory activation in a mouse model of AD [81].Through reducing glial cell inflammatory activity and enhancing glial viability, incretin-based multi-agonists may allow for reparative functions of glial cells to combat neuroinflammation and neurodegeneration.
The benefit of incretin-based multi-agonists as treatments for neurodegenerative disease lies not only in their pleiotropic therapeutic mechanisms, but also in the relative efficiency of repurposing an existing FDA-approved drug class, as compared to developing entirely new therapeutics.The safety profiles of GLP-1R single agonists as well as dual GLP-1R/GIPR agonists, namely Tirzepatide/Mounjaro ® (LY329), and triple GLP-1R/GIPR/GcgR agonists, namely Retatrutide (Triagonist), have already been established in humans, thereby saving significant amounts of time and resources.Considering the urgency of the demand for neurodegenerative disease treatments, the efficiency that repurposing incretin mimetics offers is immensely valuable.
However, an important consideration with repurposing incretin-based compounds to treat diseases of the brain is drug administration.GLP-1, GIP, and Gcg are large peptide compounds, and synthetic versions of these compounds with additional chemical mod-ifications can be even larger.As a result, limited BBB penetration is an area of concern with this class of drugs, particularly considering that the drugs are administered peripherally [94,95].Further research that investigates mechanisms of drug entry into the brain as well as pharmacokinetic studies with the more novel incretin-based multi-agonists will certainly be valuable in identifying translatable doses relevant to human neurodegenerative disease treatment.
The development and emergence of incretin-based multi-agonists on the market for T2DM and obesity treatment are quite encouraging.Although Tirzepatide/Mounjaro ® is the only currently FDA-approved multi-agonist, several additional incretin-based dual or triple agonists are in clinical trials for T2DM and obesity, including the triple agonist (Retatrutide) used in this study [96].Tirzepatide/Mounjaro ® has already demonstrated enhanced efficacy in T2DM treatment relative to single GLP-1R agonists [48].In much the same way, in our present study, multi-agonists magnified anti-neuroinflammatory and neuroprotective benefits as compared to a single GLP-1R agonist in cell culture models of neurodegenerative disease.In this regard, three different immortal CNS cell lines were evaluated in the present study to determine whether incretin-mediated actions translate across different cell types (neuronal vs. microglial), as well as across rodent and human derived cells (IMG and HMC3 microglia, respectively).Future in vivo studies and clinical trials are certainly warranted to determine whether incretin receptor multi-agonists may outperform single GLP-1R agonists as a novel therapeutic option for human neurodegenerative diseases.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biom14070872/s1,The Supplementary Materials include the original Western blots.

Figure 3 .
Figure 3. Antiapoptotic effects of incretin-based single, dual, and triple receptor agonists in neuronal cells.SH-SY5Y cells were pre-treated for 24 h with vehicle or 100 nM exendin-4 (A), Twincretin (B), or Triagonist (C), followed by a 24 h 100 or 250 nM STS pro-apoptotic challenge treatment (n = 3-4 per treatment group).Cytotoxicity levels were quantified using an LDH assay as per the kit instructions and were normalized to total protein level as determined by BCA.Values are expressed relative to Control levels.* indicates a significant difference between STS alone and STS + agonist treatment at p < 0.05, ** at p < 0.01, **** at p < 0.0001 by one-way ANOVA with Tukey's multiple comparisons test.

Figure 3 .
Figure 3. Antiapoptotic effects of incretin-based single, dual, and triple receptor agonists in neuronal cells.SH-SY5Y cells were pre-treated for 24 h with vehicle or 100 nM exendin-4 (A), Twincretin (B), or Triagonist (C), followed by a 24 h 100 or 250 nM STS pro-apoptotic challenge treatment (n = 3-4 per treatment group).Cytotoxicity levels were quantified using an LDH assay as per the kit instructions and were normalized to total protein level as determined by BCA.Values are expressed relative to Control levels.* indicates a significant difference between STS alone and STS + agonist treatment at p < 0.05, ** at p < 0.01, **** at p < 0.0001 by one-way ANOVA with Tukey's multiple comparisons test.Biomolecules 2024, 14, 872 9 of 20

Figure 4 .
Figure 4. Pro-apoptotic caspase-3 activity is reduced with Triagonist treatment.SH-SY5Y cells were pre-treated with the vehicle or 100 nM Triagonist for 24 h; 250 nM STS was subsequently added for an additional 24 h (n = 4 per treatment group).Caspase-3 activity was determined using a Fluorimetric Caspase 3 Assay Kit as per the kit instructions.Of note, Triagonist was the only incretinbased compound that we found to be effective in mitigating caspase-3 activity.Values are expressed relative to Control levels.*** indicates a significant difference at p < 0.001 by one-way ANOVA with Tukey's multiple comparisons test.

Figure 4 .
Figure 4. Pro-apoptotic caspase-3 activity is reduced with Triagonist treatment.SH-SY5Y cells were pre-treated with the vehicle or 100 nM Triagonist for 24 h; 250 nM STS was subsequently added for an

Figure 5 .
Figure5.Wide-ranging anti-neuroinflammatory abilities of incretin receptor single and multi-agonists.(A) Multi-agonists enhance IMG cell viability and, notably, reduce pro-inflammatory microglial signaling, nitrite production, and cytokine release (following normalization to cell viability).TNF-α (B), IL-6 (C), and PGE2 (F) levels in culture media were determined using corresponding ELISA kits; nitrite (NO2 − ) levels (D) in culture media were determined using a Griess reagent system nitrite assay kit; and intracellular COX2 expression (E) was determined using Western blotting and was normalized to GAPDH expression (representative bands are shown in (E) (bottom), all blots are provided in the Supplemental Materials).Mouse IMG cells were pre-treated with 100 nM exendin-4, Twincretin, LY329, or Triagonist for 24 h, followed by a 24 h 1 ng/mL LPS pro-inflammatory treatment.Cytokine, nitrite, and PGE2 levels were normalized to cell viability as determined via MTS assays (A).In (A), values are expressed relative to Control levels; in (B-F), values are expressed relative to 1 ng/mL LPS levels.(A-D) Control n = 19-23, Vehicle + LPS n = 20-24, each peptide + LPS n = 5-6; (E) Control n = 6, Vehicle + LPS n = 6, each peptide + LPS n = 3; (F) Control n = 4, Vehicle +

Figure 7 .
Figure 7. Cycle of neuroinflammation and sites of incretin-based compound anti-inflammatory activity (generated from the present studies and the scientific literature[46]).CNS stressors relevant

Figure 7 .
Figure 7. Cycle of neuroinflammation and sites of incretin-based compound anti-inflammatory activity (generated from the present studies and the scientific literature[46]).CNS stressors relevant to neurodegenerative disease (A) can activate microglia into a pro-inflammatory phenotype (B).Activated microglia release various pro-inflammatory factors, including cytokines, chemokines, prostaglandins, and other inflammatory molecules, that can bind neurons (D) and activate additional microglia (C).Damaged and degenerating neurons (E) secrete signaling molecules such as damageassociated molecular patterns (DAMPs), cell debris, ATP, and ROS that in turn activate microglia (F), fueling the neuroinflammatory cycle.Incretin-based peptides (green) can act at several of these stages to mitigate neuroinflammation-incretins can dampen microglial activation, reduce pro-inflammatory cytokine release, mitigate oxidative stress, and promote neuronal viability.Figure adapted fromKopp et al., 2022 [46].

Author Contributions:
Conceptualization, N.H.G.; Methodology, E.J.G. and D.T.; Formal analysis, Y.L.; Investigation, K.O.K. and Y.L.; Resources, E.J.G. and D.T.; Data curation, K.O.K.; Writing-original draft, K.O.K.; Writing-review & editing, Y.L., E.J.G., D.T. and N.H.G.; Visualization, K.O.K.; Supervision, Y.L.; Project administration, N.H.G.; Funding acquisition, N.H.G.All authors have read and agreed to the published version of the manuscript.Funding: This research was supported by the Intramural Research Programs of the National Institute on Aging (AG000333) and National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA.